Project Summary Pancreatic endocrine cells, including insulin-producing beta cells, acquire their fate in a step-wise manner during embryonic development. Understanding and recapitulating these steps has proven essential for directed differentiation of ES cells into beta cells. A number of academic and biopharma groups have reported improved efficiency of beta cell generation upon shifting culture methods from 2D to 3D cultures. This observation suggests that architecture of the cellular niche for beta cell generation is critical, however, this idea currently remains unexplored. We previously reported that when the pancreas first emerges, the endodermal epithelium undergoes transient stratification, followed by microlumen formation and fusion to generate a 3D network of interconnected epithelial tubes called the pancreatic `plexus' [1]. Interestingly, recent studies demonstrate that endocrine progenitors are born within this transient core plexus. It is unclear how the plexus architecture impacts the fate of pancreatic progenitors, including those of endocrine lineage. Since our initial proposal, we published the findings that Afadin is essential to pancreas morphogenesis and endocrine fate (Azizoglu et al., 2017). Here, we propose to elucidate the cellular and molecular mechanisms by which Afadin controls epithelial lumen formation and plexus morphogenesis. In previous work, we generated a mutant mouse with deletion of the junctional and cytoskeletal regulator Afadin (AfapancKO) that fails to resolve its transient plexus. Co-depletion of Afadin and RhoA (AfaRhoApancKO or AfaRhoDKO) exhibits multiple lumen defects. Surprisingly, it also produces an increase in endocrine cell numbers, including beta cells. RhoApancKO however, show no pancreatic defects. How Afadin and RhoA pathways interact remains unknown. One striking observation in both AfapancKO and AfaRhoDKO is that the core plexus persists. We propose that the progenitor pool and final endocrine mass is determined by the perdurance of the core plexus. How this occurs is the central question of this proposal. We hypothesize that Afadin and RhoA drive epithelial lumen morphogenesis (formation/extension/resolution) via regulation of cellular processes, such as vesicle trafficking (Aim 1), and cell division and/or cell migration (Aim 2). Further, we hypothesize that Afadin and RhoA control these processes by regulating cytoskeletal organization (Aim 3). Together, these processes coordinate to build a niche propitious for generation of endocrine cells. Completion of these studies will expand our knowledge of pancreatic development, and will lead to enhanced strategies for generating endocrine cells, including beta cells, which may contribute to novel treatments for type I diabetic patients.